Plants can detect thousands of different pathogens and actively respond. It is not understood how this detection is accomplished, or how detection is translated into a resistance response. The proposed research addresses these questions using genetics and protein biochemistry. We have identified and isolated the Arabidopsis disease resistance (R) gene RPS5, which mediates recognition of the AvrPphB protein secreted by the bacterial pathogen Pseudomonas syringae. RPS5 belongs to the largest class of plant R gene products, which is defined by the presence of a putative nucleotide bindings site (NB) and leucine rich repeats (LRRs). NB-LRR proteins are also found in humans, and at least two have been shown to activate immune responses upon detection of bacterial cell wall fragments. Studies of plant NB-LRR proteins will likely provide insights into the functions of mammalian NB-LRR proteins, and vice versa. A specific goal of the proposed research is to identify the mechanism by which RPS5 detects AvrPphB and activates defense responses in plants. Current work in the Innes laboratory has shown that AvrPphB is a cysteine protease that specifically targets the Arabidopsis PBS1 kinase, which appears to regulate defense genes. Cleavage of PBS1 is required for activation of RPS5, but how this cleavage event activates RPS5 is unknown. Based on analogies to human NB-LRR proteins, cleavage of PBS1 may produce a ligand that binds to RPS5, which then induces oligomerization of RPS5 via the NB domain. This model will be tested using both in vitro and in vivo (plant and yeast) binding assays to test for physical interactions between PBS1 and RPS5, and between RPS5 and itself. Multiple derivatives of each will be tested for binding activity in order to identify specific subdomains and phosphorylation states that are critical to RPS5 activation. In addition, the defense pathways regulated by PBS1 will be identified using Affymetrix GeneChip analysis, and putative substrates of PBS1 will be identified using a split ubiquitin yeast two-hybrid screen.